www.nature.com/scientificreports

OPEN Genome‑wide analysis of RING‑type E3 ligase family identifes potential candidates regulating high amylose starch biosynthesis in wheat (Triticum aestivum L.) Afsana Parveen1,2, Mohammed Saba Rahim1, Ankita Sharma3, Ankita Mishra1, Prashant Kumar1, Vikas Fandade1, Pankaj Kumar1, Abhishek Bhandawat1, Shailender Kumar Verma3 & Joy Roy1*

In ubiquitin-mediated post-translational modifcations, RING fnger families are emerged as important E3 ligases in regulating biological processes. Amylose and amylopectin are two major constituents of starch in wheat seed endosperm. Studies have been found the benefcial efects of high amylose or resistant starch on health. The ubiquitin-mediated post-translational regulation of key enzymes for amylose/amylopectin biosynthesis (GBSSI and SBEII) is still unknown. In this study, the genome- wide analysis identifed 1272 RING domains in 1255 proteins in wheat, which is not reported earlier. The identifed RING domains classifed into four groups—RING-H2, RING-HC, RING-v, RING-G, based on the amino acid residues (Cys, His) at metal ligand positions and the number of residues between them with the predominance of RING-H2 type. A total of 1238 RING protein genes were found to be distributed across all 21 wheat chromosomes. Among them, 1080 RING protein genes were identifed to show whole genome/segmental duplication within the hexaploid wheat genome. In silico expression analysis using transcriptome data revealed 698 RING protein genes, having a possible role in seed development. Based on diferential gene expression and correlation analysis of 36 RING protein genes in diverse (high and low) amylose mutants and parent, 10 potential RING protein genes found to be involved in high amylose biosynthesis and signifcantly associated with two starch biosynthesis genes; GBSSI and SBEIIa. Characterization of mutant lines using next-generation sequencing method identifed unique mutations in 698 RING protein genes. This study signifes the putative role of RING-type E3 ligases in amylose biosynthesis and this information will be helpful for further functional validation and its role in other biological processes in wheat.

Bread wheat (Triticum aestivum) is a rich source of starch (~ 70%)1, that comprising of amylose (~ 25%) and amylopectin (~ 75%)2. High amylose starch has nutritional benefts as it is classifed under type-2 resistant starch (RS) category. Te resistant starch is slowly digestible in human gut, therefore it has low glycemic index. It is benefcial for combating gastrointestinal diseases, diabetes and obesity­ 3–6. In wheat, the natural variation for high amylose starch is narrow. It can be improved by genetic engineering tools using the key pathway genes. It is found that the progress is limited as overexpression and knock-down/out of two key genes—GBSSI and SBEII has limited ­success7–9. Tis can also be done by mutational breeding ­approaches10. Many post translational studies disclosed the diferent aspects of starch biosynthesis pathway­ 11–13 to date but the ubiquitin-mediated post

1Agri‑Food Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali 140306, Punjab, India. 2Department of Biotechnology, Panjab University, Chandigarh 160014, Punjab, India. 3Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh, Kangra 176206, Himachal Pradesh, India. *email: [email protected]

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 1 Vol.:(0123456789) www.nature.com/scientificreports/

translational insights of the starch biosynthesis pathway has not yet revealed and no attempt has been done for high amylose starch. Ubiquitin-mediated post-translational modifcations targeting protein degradation has emerged as a crucial process in controlling diferent aspects of cellular processes in eukaryotes­ 14,15. Tis process involves the subse- quent action of three enzymes, E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligase)16. In this multistep process E3 ubiquitin ligases are key determinant of target specifcity for degradation by 26S proteasome ­system17,18, which are mainly classifed in two groups HECT (Homologous to the E6-associated protein C terminus) and RING (Really Interesting New Gene) fnger/U-box ­domain17,19. Of these known E3 ligases, RING fnger domain containing proteins comprise major proportion. Te RING domains are considered to be involved in protein–protein interactions and essential for E2 dependent ubiquitination, that can function as a single subunit or in multi-subunit complexes­ 20–22. RING (Really Interesting New Gene/U-box)-type E3 ligases belong to the largest class of E3 ligases with > 600 members in human as the RING E3 ligases function is diverse and act as allosteric activators of the E2. Initially the RING domains were characterized as RING-H2 and RING-HC type on the basis His and Cys at ffh metal ligand position, respectively. Further 5 modifed RING types RING-C2, RING S/T, RING-v, RING-D and RING-G having variation in amino acid residue at metal ligand positions were also identifed in A. thaliana23,24. Te role of RING fnger E3 ligases in the plant development have been extensively studied for various bio- logical processes. RING fnger containing proteins like COP1 is a well-known for its role as photomorpho- genic repressor­ 25, XBAT35.2 in cell death induction and pathogen response­ 26, HOS1 in cold response­ 27, KEG in growth and ­development28, Capsicum annuum CaAIRF1 in ABA and drought ­signaling29, ATL2/ATL9 in defense ­response30. Recent studies revealed that SP1, a RING type E3 ligase is involved in degradation of TOC compo- nents (translocon) present in chloroplast outer membrane and hence regulate the chloroplast protein import­ 31. However, the role of this degradation pathway in amyloplast proteins turnover remains unclear. Beside the role in diferent biological processes the RING fnger E3 ligase GW2 found to be involved in grain size and weight in rice and wheat possibly by regulating the expression of starch biosynthetic pathway genes­ 32–34. Te reduction in the transcripts of GW2 by RNAi in the durum wheat cultivar increased the starch content by 10–40%, width by 4–13% and surface area by 3–5%, that suggest the active role of GW2 RING fnger E3 ligase in starch biosynthesis but its interacting partners need to be explored­ 35. Tese previous studies provides substantial information to perform the current study. In this study, we reported the genome-wide identifcation and characterization of RING domain containing E3 ligase family in wheat as well as identifed the putative RING E3 ligases that may involve in high amylose biosynthesis based on quantitative gene expression and correlation analysis in developing wheat grains. A large set of 1255 proteins containing 1272 RING domains was frst time identifed in wheat and 10 potential RING protein genes found to be involved in high amylose biosynthesis and signifcantly associated with two starch biosynthesis genes; GBSSI and SBEIIa. Further, the transcriptome sequencing using next-generation sequenc- ing method identifed several unique induced mutations in 698 RING protein genes. Hence, this studies lay the foundation for future research to make better understanding of RING fnger E3 ligases involvement in high amylose starch biosynthesis in cereal crops. Results Identifcation and classifcation of RING fnger proteins in wheat. A total of 1272 potential RING domains in 1255 proteins were identifed in wheat proteome through in silico studies (Supplementary Table S1). Among the identifed proteins, 1241 proteins contained a single RING domain, 12 proteins with two RING domains and one protein each with three and four domains. Proteins with multiple RING domains were suf- fxed with an alphabet to their gene IDs. Predicted RING domain containing proteins size ranged from 84 to 4749 amino acids with domain size ranging from 30 to 102 amino acids. On the basis of amino acid residues at eight metal ligand positions (Cys and/or His) and number of residues between them, 1272 RING domains were classifed into 4 groups according to Stone et al., (2005). Maximum number of RING domains i.e. 875 domains (68.79%) were identifed as RING-H2 followed by 323 RING-HC domain (25.39%), 67 RING-v domains (5.27%) and 7 RING-G domains (0.55%) (Supplementary Table S1, Supplementary Fig. S1). Representative sequence logos of protein motifs of the four identifed groups are shown in Fig. 1. In RING-H2 type (out of 875) six domains were identifed as RBX type having Asp instead of Cys at eighth metal ligand position (Supplementary Fig. S1). In wheat we did not identify RING-D, RING-C2 and RING-S/T type domains which were present in Arabidopsis, Brassica rapa, apple and rice. In this study, a large number of RING containing proteins were identifed as compared to other plant species such as Arabidopsis (469), rice (488), apple (663) B. rapa (715) and B. oleracea (734) 24,36–39. Additionally, 117 RING domains in 94 proteins were also identifed by in silico studies but due to the absence or substitution of one or more amino acid residues at metal ligand positions they were not classifed in any group and considered as incomplete RING domains (Supplementary Table S2). But for the downstream analysis only complete RING protein genes were considered.

Phylogenetic analysis. To expand the knowledge on wheat RING proteins, this study included phylo- genetic analysis among 1272 RING domain sequences of 1255 wheat RING proteins. Majority of similar type of RING domains (RING-H2, RING-HC, RING-v and RING-G) were clustered together with various small subgroups (Supplementary Fig. S2). Te phylogenetic relationship suggested that most of the domains from the same group were categorized together while some were found intermixed with other domains. Tis topology might be due to the discrepancy in domain sequences except the conserved metal ligands, during evolutionary ­process40. RING-H2 and RING-HC domains tend to be distributed within each other forming several subgroups

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 2 Vol:.(1234567890) www.nature.com/scientificreports/

Figure 1. Sequence logo of overrepresented motifs found in RING-H2, RING-HC, RING-v, RING-G domains, respectively generated using WebLogo program version 2.8.2 (https://weblo​ go.​ berke​ ley.​ edu/​ logo.​ cgi​ ). Te fgures were generated by on-line WebLogo. Te height of the letters indicates the conservation at that particular position. Te asterisked letters indicate the conserved metal ligands and zinc coordinating amino acid pairs.

containing small number of domains. Te RING-G and RING-v domains were clustered together but lied within major RING-H2 and RING-HC domain groups.

Additional domains in RING fnger proteins. Out of the 1255 RING fnger proteins, 885 proteins (70.52%) contained one or more additional domains other than RING (Supplementary Table S3). On the basis of presence/absence and organization of the additional domains, the 1255 RING domain containing proteins were grouped into 36 major groups (Table 1; Supplementary Table S3). Of 885 RING domain containing pro- teins, 544 (group 6.1) proteins contained additional transmembrane domain (1–14 repetitions) and 43 (group 3) proteins had only coiled coil (repetition 1–6) domain. Te Zinc-Ribbon-9, signal peptide and VWA domains were present in 26, 19 and 18 RING domain containing proteins of group 13.1, 21.1 and 11.1 respectively. Some of the RING fnger proteins were found to have two or more other domains along with the aforementioned domains. As with the transmembrane domain other domains, namely GIDE, IBR, PA, VWA, zinc_ribbon_9, Tmemb_185A, coiled-coil and signal peptide were also present in RING domain containing proteins. A protein– protein interacting domain VWA was simultaneously found in 20 RING proteins in combination with Vwaint domain. Te Vwaint domain previously not found in Arabidopsis RING fnger ­proteins24 but further identifed in ­brassica39. Te two domains Copine and NB-ARC were specifcally found in wheat RING proteins, previously not found in any other plant species. Four domains ZnF_UBP, GIDE, RWD and CUE involved in ubiquitina- tion were also identifed. Nucleic acid binding domains DEXDc, HELICc, HIRAN, ZNF_C2H2, ZNF_C3H1 and ZNF_C2HC were identifed in 27 RING fnger proteins. Protein–protein interaction domains, Ankyrin repeats, BRCT, TPR, VWA, Coiled-coil and WD40 repeats were identifed in RING fnger proteins that help in substrate recognition by E3 ligases­ 41–43 Te two protein–protein interaction domains PHD and ZnF_ZZ from the same family of cross-brace zinc fnger, from which RING domain belongs, were also identifed. Presence of these various types of additional domains indicates that instead of RING fnger domains these proteins share diferent features and are very distinct to each other.

Chromosomal locations and duplication events analysis. Te chromosomal location of 1255 RING protein genes were extracted from T. aestivum genome database downloaded from Ensembl plants (fp://​fp.​ Ensem​blgen​omes.​org/​pub/​plants/​relea​se-​46/​gf3/​triti​cum_​aesti​vum). Out of 1255, 17 genes were mapped to

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 3 Vol.:(0123456789) www.nature.com/scientificreports/

Sr. no. Group no. No. of proteins Representative gene IDs Additional domain/s Species* Domain description RING domain, protein binding, At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 1 1 370 TraesCS1A02G090900 RING Zinc ion binding, E3 ubiquitin Mm, Sc ligase ­activity20 Ankyrin repeats; protein–protein 2 2 3 TraesCS1A02G088100 ANK At, Ce, Dr, Gm, Hs, Mm ­interactions61 3 3 43 TraesCS1A02G084200 Coiled-coil At, Dd, Dr, Gm, Hs, Mm, Pf Coiled-coil domain BRCA1-associated protein ­262, At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, Ubiquitin C-terminal hydrolase- 4 4.1 2 TraesCS6A02G145300 BRAP2, ZnF_UBP Mm, Pf, Sc, Sp like zinc fnger; ubiquitinbinding domain At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 5 4.2 1 TraesCS6D02G134300 Coiled-coil, BRAP2, ZnF_UBP See group 3, See group 4.1 Mm, Pf, Sc, Sp Ca(2+)-dependent phospholipid- 6 5 1 TraesCS5B02G111900 Copine At, Ce, Dd, Dm, Dr, Gm, Hs, Mm binding ­proteins63 At, Ce, Ce, Dd, Dm, Dr, Gm, Hs, 7 6.1 544 TraesCS1A02G126200 Transmembrane Transmembrane Mm, Sc 8 6.2 6 TraesCS7B02G442100 Transmembrane, coiled-coil At, Dd, Dm, Dr, Gm, Hs, Mm See group 6.1, See group 3 IBR, In between ring fngers, At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 9 7.1 8 TraesCS2A02G546000 IBR cysteine-rich (C6HC) zinc fnger Mm, Sp domain At, Ce, Dd, Dm, Dr, Gm, Hs, 10 7.2 1 TraesCS2D02G187100 IBR, Signal peptide See group 7.1, Signal peptide Mm, Sp See group 3, Kinesin motor domain; ATPase activity, role 11 8 3 TraesCS1A02G177600 Coiled-coil, KISc At, Ce, Dd, Dm, Dr, Gm, Hs, Mm in cell division and organelle transport Helicase superfamily c-terminal At, Cd, Ce, Dd, Dm, Dr, Gm, Hs, 12 9.1 1 TraesCS1B02G074500 HELICc domain; helicase activity; ATP Ld, Mm, Pf, Sc, Sco, So, Sp, Sp binding; nucleotide-binding64 At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, DEAD-like helicases; ATP binding 13 9.2 4 TraesCS2A02G224300 DEXDc, HELICc Mm See group 9.1 See group 9.2, See group 9.1, found At, Cd, Ce, Dd, Dm, Dr, Gm, Hs, 14 9.3 9 TraesCS2A02G145200 DEXDc, HELICc, HIRAN in N-terminal regions of SWI2/ Ld, Mm, Pf, Sc, Sp, Spn SNF2 ­ptoteins65 Plant homeodomain C4HC3 At, Gm, Dr, Mm, Hs, Dm, Pf, Ld, 15 10 1 TraesCS1D02G002400 PHD, SRA ­type66, SET and RING fnger asso- Dr, Sco ciated domain Von Willebrand factor (vWF) type 16 11.1 18 TraesCS3A02G414400 VWA At, Ce, Dd, Dr, Gm, Hs, Ld, Mm A domain 17 11.2 1 TraesCS5D02G122000 Signal peptide, VWA At, Ce, Dd, Dr, Gm, Hs, Ld, Mm See group 7.2, See group 11.1 At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 18 12 1 TraesCS3D02G079000 Coiled-coil, TPR_7 See group 3, TPR_7 Mm, Pf At, Ce, Dd, Dm, Dr, Gm, Hs, Mm, 19 13.1 26 TraesCS1A02G162400 Zinc_ribbon_9 Zinc-binding ribbon domain Pf, Sp At, Dd, Dm, Dr, Gm, Hs, Mm, 20 13.2 3 TraesCS5A02G467700 Zinc_ribbon_9, coiled-coil See group 13.1, See group 3 Pf, Sp At, Ce, Dd, Dm, Dr, Gm, Hs, 21 13.3 2 TraesCS7A02G169600 Zinc_ribbon_9, transmembrane See group 13.1, See group 6.1 Mm, Pf Signalling motif found in bacteria 22 14 1 TraesCS7A02G005100 NB-ARC​ At, Gm and ­eukaryotes67 Domain of unknown function DUF4792, DUF4793, signal approximately 70 residues, Domain 23 15 3 TraesCS1A02G341400 At, Dd, Dm, Dr, Gm, Hs, Ld, Mm peptide of unknown function approxi- mately 110 residues, See group 7.2 Domain of unknown function, See 24 16 8 TraesCS1A02G029800 DUF1117, Zinc_ribbon_9 At, Dd, Dm, Dr, Gm, Hs, Mm, Pf group 13.1 At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 25 17.1 1 TraesCS2B02G577000 IBR, transmembrane See group 7.1, See group 6.1 Mm, Sp IBR, signal peptide, transmem- At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, See group 7.1, See group 7.2, See 26 17.2 1 TraesCS2D02G547300 brane Mm, Sp group 6.1 ATP-dependent protease La (LON) At, Ce, Dm, Dr, Gm, Hs, Mm, Pa, 27 18 3 TraesCS4A02G162000 LON_substr_bdg domain; ATP- dependent Ser Pf, Sh, Sp ­peptidases68 At, Ce, Dd, Dm, Dr, Gm, Hs, Mm, 28 19 9 TraesCS4A02G117900 PHD See group 10 Pf, Sp 29 20.1 1 TraesCS7B02G226400 PA, TRANSMEMBRANE At, Ce, Dm, Dr, Gm, Hs, Mm See group 6.1 30 20.2 5 TraesCS2B02G625300 PA, signal peptide, transmembrane At, Ce, Dm, Dr, Gm, Hs, Mm, Sp See group 7.2, See group 6.1 31 21.1 19 TraesCS1B02G156500 Signal peptide At, Ce, Dm, Dr, Gm, Hs, Mm See group 7.2 Signal peptide, TRANSMEM- At, Ce, Dd, Dm, Dr, Gm, Hs, Mm, 32 21.2 43 TraesCS2B02G336300 See group 7.2, See group 6.1 BRANE Sp, Sp Continued

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 4 Vol:.(1234567890) www.nature.com/scientificreports/

Sr. no. Group no. No. of proteins Representative gene IDs Additional domain/s Species* Domain description Signal peptide, transmembrane, See group 7.2, See group 6.1, See 33 21.3 2 TraesCS3A02G051000 At, Gm coiled-coil group 3 At, Ce, Dd, Dm, Dr, Gm, HS, Ld, 34 22 6 TraesCS1A02G025700 PHD, BRCT​ See group 10 Mm, Sp 35 23 3 TraesCS2A02G094000 PHD, coiled-coil, AT_hook At, Dd, Dr, Gm, Hs, Mm See group 10, See group 3 Zinc-ribbon fnger, each pair of At, Ce, Dd, Dm, Dr, Gm, Hs, Mm, 36 24.1 2 TraesCS1A02G143300 Zinc_ribbon_6 zinc-ligands coming from more-or- Pf, Sc, Sp less either side of two ­knuckles69 At, Ce, Dd, Dm, Dr, Gm, Hs, Pf, See group 24.1, Zinc ion binding; 37 24.2 8 TraesCS1D02G142100 Zinc_ribbon_6, zf-CHY Sc, Sp function ­unknown70 See group 24.1, See group 24.2, Zinc_ribbon_6, zf-CHY, Hem- At, Ce, Dd, Dm, Dr, Gm, Hs, 38 24.3 3 TraesCS1A02G374800 responsible for oxygen (O2) trans- erythrin Mm, Sp port in the marine invertebrate C2H2-type zinc fnger, binds to 39 25 1 TraesCS3A02G288900 ZnF_C2H2 At, Sp, Dd, Dm, Dr, Gm, Hs, Mm DNA, RNA and protein targets Zinc-fnger domain At, Hs, Ce, Dd, Dm, Dr, Gm, Ld, 40 26 9 TraesCS4A02G082400 ZnF_C3H1 C- × 8-C- × 5-C- × 3-H type; nucleic Mm, Pf, Sc, Sp acid ­binding71 At, Dd, Dm, Dr, Gm, Hs, Mm, Zinc-fnger domain 41 27 3 TraesCS6B02G255500 ZnF_ZZ Pf, Sp C- × 2-C- × 5-C- × 2-C ­type72 At, Ce, Dd, Dm, Dr, Gm, Hs, Mm, See group 7.1, function in protein 42 28 3 TraesCS2A02G446100 IBR, RWD, ZnF_C2HC Sc, Sp interaction, See group 6.1 43 29.1 1 TraesCS1B02G007900 SRA, coiled-coil At, Dr, Dra, Gm, Hs, Mm, Sc, Sc See group 10, See group 3 See group 3, See group 10, See 44 29.2 1 TraesCS1A02G005700 Coiled-coil, PHD, SRA At, Gm, Mm, Hs, Dr, Dra, Sco group 10 At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, Transmembrane Fragile-X-F pro- 45 30 6 TraesCS5D02G213300 Tmemb_185A Mm tein, linked to FragileXF syndrome Lies between the N-terminal VWA At, Ce, Dd, Dm, Dr, Gm, Hs, Ma, domain and the more C-terminal 46 31.1 20 TraesCS1A02G146700 Vwaint, VWA Mm, Pf, Sc, Sh, Sp ’Vint’-type Hint ­domain73, See group 11.1 At, Ce, Dm, Dr, Gm, Hs, Ld, Ma, See group 31.1, See group 7.2, See 47 31.2 3 TraesCS2A02G364100 Vwaint, Signal peptide, VWA Mm, Pf, Sc, So, Sp group 11.1 At, Dr, Gm, Hs, Ma, Mm, Sco, See group 3, See group 31.1, See 48 31.3 2 TraesCS4B02G344200 Coiled-coil, Vwaint, VWA So, Ssp. group 11.1 See group 6.1, Domain of unknown At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 49 32.1 4 TraesCS3B02G567600 Transmembrane, DUF1232 function representing a conserved Mm, Pf, Sc region of approximately 60 residues See group 6.1, Domain of unknown 50 32.2 23 TraesCS1A02G206100 Transmembrane, DUF3675 At, Ce, Dd, Dr, Gm, Hs, Mm function approximately 120 amino acids in length See group 6.1, Involved in binding At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 51 33 2 TraesCS2A02G227400 Transmembrane, CUE ubiquitin-conjugating enzymes; MM, Pf, Sc, Sp ubiquitin-binding ­motif74 Found as tandem repeats each At, Ce, Dd, Dm, Dr, Gm, Hs, Ma, containing a central Trp/Asp 52 34.1 3 TraesCS1A02G263800 WD40 Mm, Sc, Sp, Ssp. motif; Phospho-Ser/Tr-binding ­domain75 At, Ce, Dd, Dm, Dr, Gm, Hs, Ld, 53 34.2 3 TraesCS6A02G326100 WD40, coiled-coil See group 34.1, See group 3 Ma, Mm, Sc, Sp E3 ubiquitin ligase involved in 54 35 2 TraesCS2D02G141100 GIDE, signal peptide At, Dd, Dm, Dr, Gm, Hs, Ld, Mm inducing ­apoptosis76, See group 7.2 At, Ce, Dd, Dm, Dr, Gm, HS, Ld, 55 36 3 TraesCS4A02G011000 GIDE, transmembrane See group 35, See group 6.1 Mm, Pf

Table 1. Wheat RING domain containing proteins grouped based on the presence or absence and organization of the additional domain/s in RING proteins. Te possible wheat RING protein orthologs in other species are shown. *Species having similar domain architecture in RING proteins determined by NCBI BLASTp search in model organisms (landmark) database only. At: Arabidopsis thaliana, Cd: Clostridioides difcile, Ce: C. elegans, Dd: Dictyostelium discoideum, Dm: Drosophila melanogaster, Dr: Danio rerio, Dra: Deinococcus radiodurans, Gm: Glycine max, Hs: Homo sapiens, Ld: Leishmania donovani, Ma: Microcystis aeruginosa, Mm: M. musculus, Pa: Pseudomonas aeruginosa, Pf: Plasmodium falciparum, Sc: Saccharomyces cerevisiae, Sco: Streptomyces coelicolor, So: Shewanella oneidensis, Sp: Schizosaccharomyces pombe, Spn: Streptococcus pneumonia, Ssp.: Synechocystis sp., Xl: Xenopuslaevis, Zm: Zea mays.

unassembled chromosomal locations that were excluded in this study. Te retrieved chromosomal locations of rest 1238 wheat RING protein genes were mapped on wheat genome that were randomly distributed on all the 21 chromosomes (1A, 1B, 1D to 7A, 7B, 7D) (Supplementary Table S4). RING protein genes were found to be less distributed on 4B (3.5%) and largely distributed on 7D (6.7%) chromosome (Supplementary Fig. S3). Te total number of genes in each RING class (RING-H2, RING-HC, RING-v, RING-G) were also determined and

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 5 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 2. Histogram representing the distribution of RING protein genes in the identifed four RING domain groups on 21 chromosomes (Ta1A to Ta7D) based on chromosomal locations from Ensembl Triticum aestivum database. Where, Ta stand for Triticum aestivum followed by chromosome name.

it was observed that RING-H2, RING-HC, and RING-v type RING protein genes were distributed throughout all the 21 chromosomes but genes from RING-G class were only present on chromosomes 1B, 1D, 4B and 5A (Fig. 2). Average distance between two RING protein genes ranged from 7.6 to 15.6 Mb on chromosomes 1D and 4B, respectively. Te results decipher that RING protein genes are inconsistently distributed on wheat genome. Meanwhile, singleton (not duplicated) and four duplication events dispersed, proximal, tandem, WGD/segmen- tal in 4, 49, 15, 90, and 1080 RING protein genes were identifed, respectively (Supplementary Table S4). Whole genome duplicated/segmental, tandem and dispersed (except 2A and 6D) duplicated genes were distributed all over the genome. Singleton were found only on 1B, 3D, 4B, and 6D, while proximal were found on 1A, 1B, 1D, 2A, 3A, 5A, 5D, 7A and 7D. Whole genome/segmental duplicated genes from each chromosome are displayed in Fig. 3.

Expression profling during grain development. To explore the potential role of RING domain con- taining proteins during wheat grain development, gene expression values (TPM -Transcripts Per Million) of 1255 RING protein genes at diferent developmental stages i.e., 2 DAA, 14 DAA and 30 DAA was retrieved from publicly available wheat expression database. Out of the 1255 RING protein genes, 698 were found expressed at any of the three developmental stages suggesting their potential role during grain development. However, 557 genes that were not found expressed at any of the three stages, were considered as insignifcant and excluded from this study. Of the 698 RING protein genes, 306 were consistently expressed at all the developmental stages. We found three diferent patterns of expression including a combination of any two days i.e., (i) 2 and 14 DAA, (ii) 14 and 30 DAA, and (iii) 2 and 30 DAA, these three combination included 22, 3, and 156 genes respectively. Days specifc expression of the RING protein genes was also found involving 152, 5, and 54 genes expressed at 2, 14, and 30 DAA (Fig. 4). To analyze the pattern of expression of the identifed 698 RING protein genes clustered heat map was generated (Fig. 5, Supplementary Fig. S4). Based on expression pattern genes were clustered into four major groups (Group I, II, III and IV) with two subgroups: Group III (III A, III B) and Group IV (IV A, IV B) (Fig. 5). Group I included majority of genes (123) with high expression levels at 2 DAA. A total of 194 genes with almost same expression patterns at 2 DAA and 30 DAA were clustered in group II. Group III A represented 167 genes that were mainly expressed at 30 DAA compared to 2 DAA. Simultaneously, Group III B included 66 genes that were expressed at 14 and 30 DAA but with lower expression values. Group IV A contained the 133 genes expressed at 2 DAA and 14 DAA and IV B included the 15 genes particularly expressed at 14 DAA. It was observed from this expression analysis majority of RING containing protein genes were having higher expres- sion levels at initial (2 DAA) and late stages (30 DAA) of seed development in comparison to mid stages (14 DAA). Tis information briefs the involvement of RING protein genes in all stages of seed development and its functional diversity. Further to examine the putative role of the 698 RING protein genes in amylose biosynthesis during grain development, diferential gene expression (DEGs) analysis was performed using the In-house transcriptome

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 6 Vol:.(1234567890) www.nature.com/scientificreports/

Figure 3. Chromosomal locations and whole genome/segmental duplication events of 1238 RING protein genes on 21 chromosomes of hexaploid wheat. Te sufx afer ‘chr’ represent wheat chromosome name (chr1A to chr7D) and is given a diferent color.

Figure 4. Venn diagram showing number of commonly and uniquely expressed RING fnger containing genes at three seed developmental stages (2, 14 and 30 DAA). Te data were taken from the publicly available RNA- seq databases of wheat (https://wheat.​ pw.​ usda.​ gov/​ GG3/​ node/​ 237​ ). DAA stands for day afer anthesis.

data of high (‘TAC 75’) and low (‘TAC 6’) amylose mutant lines along with parent (‘C 306’). Te DEGs were ana- lyzed in two groups: Group 1 (‘TAC 75’ vs ‘C306’), and Group 2 (‘TAC 6’ vs ‘C306’). Te DEGs were considered

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 7 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 5. Expression profles of 698 wheat RING protein genes at three seed developmental stages (2, 14, and 30 day afer anthesis, DAA). Te data was extracted from RNA-seq data set (https://wheat.​ pw.​ usda.​ gov/​ GG3/​ ​ node/237​ ), clustered expression heat map generated by MeV sofware version 4.9.0 and divided into four major groups according to gene expression patterns represented as color gradient. Te color scale indicating largest gene expression values in pink color, intermediate values in black color and the smallest values in green color.

signifcantly expressed following the criteria of ­log2FC > 1 and p-value ≤ 0.05. Under this criteria ffeen genes were found signifcant in Group 1 (nine) and Group 2 (six) respectively (Supplementary Table S5). Two DEGs TraesCS6B02G286500 and TraesCS1A02G206100 in Group 1 (‘TAC 75’ vs ‘C306’) showed signifcant (seven fold) up- and down-regulated expression respectively. While, the DEG TraesCS2A02G099300 showed signifcant 11 fold down-regulated expression in group 2 (‘TAC 6’ vs ‘C306’). Te above 15 diferentially expressed RING proteins genes in group 1 and group 2 might have potential role in amylose biosynthesis. So to predict the involvement of RING proteins in amylose biosynthesis all 15 DEGs were further validated by qRT-PCR in amylose mutant lines (‘TAC 75’ and ‘TAC 6’) and parent variety (‘C 306’). Considering the non-ubiquitous expression of the RING protein genes throughout the grain development, addi- tional 21 highly expressed RING protein genes previously mined from the publicly available database (http://​ www.​wheat-​expre​ssion.​com/) were also considered for qRT-PCR based expression analysis.

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 8 Vol:.(1234567890) www.nature.com/scientificreports/

Number of variants Variant type ‘TAC 75’ ‘TAC 6’ 3 prime UTR​ 18 17 5 prime UTR premature start codon gain 1 2 5 prime UTR​ 2 14 Conservative inframe deletion 0 1 Disruptive inframe insertion 1 1 Disruptive inframe insertion & splice region 1 0 Downstream gene 32 48 Frameshif 3 4 Frameshif & splice region 0 1 Intergenic region 133 191 Intron 53 105 Missense 18 24 Missense & splice region 1 0 Splice acceptor & intron 16 9 Splice donor & intron 7 7 Splice donor & splice region &intron 0 2 Splice region & intron 19 18 Synonymous 33 35 Upstream gene 119 188

Table 2. Total number of variants identifed in diferent variant types in mutant lines ‘TAC 75’ and ‘TAC 6’.

Analysis and characterization of RING protein gene variants in mutant lines. Diferent geno- typic variants were identifed in ‘TAC 75’ and ‘TAC 6’ in reference to parent variety (‘C 306’), randomly distrib- uted across the wheat genomes (A, B and D). Tis analysis revealed a total of 457 and 667 variants in 202 and 250 RING protein genes in ‘TAC 75’ and ‘TAC 6’ respectively. Highest number of mutations were located on chro- mosome 2B and least on 4D in both mutants. Mutations including single nucleotide polymorphisms (SNPs), insertion-deletions (InDels), and multiple nucleotide polymorphisms (MNPs) at diferent positions involving genic, intergenic, and 3′ and 5′ UTR were identifed. Te predicted efect of identifed variants was characterized into high, low, moderate and modifer variants, in which 26 mutations in ‘TAC 75’ and 23 in ‘TAC 6’ exhibited high variant efect (Supplementary Table S6). Largest number of mutations were identifed within intergenic regions (29.1% and 28.64% in ‘TAC 75’ and ‘TAC 6’ respectively) followed by upstream gene variants (26.04% and 28.19% in ‘TAC 75’ and ‘TAC 6’ respectively) and intronic variants (11.6% and 15.7% in ‘TAC 75’ and ‘TAC 6’ respectively), hence amino acids remained unaltered. Conservative, disruptive, splice, synonymous, missense and frameshif variants were found comparatively lower in both ‘TAC 75’ and ‘TAC 6’ (Fig. 6), but some with deleterious efect on proteins. Te detail of total number of identifed variants listed in Table 2. Mutant charac- terization indicated the presence of EMS induced specifc mutations in RING protein genes identifed in high (‘TAC 75’) and low (‘TAC 6’) mutant lines making them unique for the variants. One of the previous studies from our ­lab6, has also characterized the two mutant lines for key enzymes GBSSI and SBEII responsible for amylose biosynthesis and possessing the defned amylose contents.

qRT‑PCR validation of candidate genes during seed development. A total of 36 RING protein genes, 15 genes from In-house transcriptome data and 21 from publicly available wheat expression database were considered for qRT-PCR validation along with two key regulators (GBSSI and SBEIIa) for amylose bio- synthesis. Information of gene specifc primers used in the study is given in Supplementary Table S7. Gene expression analysis was performed in high amylose (‘TAC 75’), low amylose (‘TAC 6’) mutant lines and parent variety (‘C 306’) at four developmental stages (7, 14, 21 and 28 DAA) to possibly cover all stages of endosperm development. Diferential gene expression was analyzed in two combinations, Group 1 (‘TAC 75’ vs. ‘C 306’), and Group 2 (‘TAC 6’ vs. ‘C 306’). All the 36 RING protein genes showed the variable pattern of diferential expression among three groups (Supplementary Table S8, Fig. 7). Diferential gene expression analysis showed that most of the RING protein genes were down-regulated in high amylose line, when compared to parent (Group 1). Out of the 36 RING protein genes, eight genes showed consistent down-regulated expression in high amylose line at all four developmental stages (Fig. 7). Out of the above eight RING protein genes, four genes (TraesCS6D02G254700, TraesCS2D02G162600, TraesCS3B02G000800, TraesCS5A02G049400) showed signifcant down-regulated expression (log­ 2 FC > 2) at the later stages (21 and 28 DAA) of seed development. However the remaining four genes (TraesCS6B02G301900, TraesCS3B02G461900, TraesCS6D02G278100, TraesCS7B02G118700), were found signifcantly down-regulated during the early stages (7 and 14 DAA) of seed development (Fig. 8). Tese results suggest that the down-regulated expression of the above identifed eight RING protein genes in high amylose lines, might play role in negative regulation of high amylose biosynthesis.

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 9 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 6. Percent distribution of diferent genetic variants in mutant lines ‘TAC 75’ and ‘TAC 6’.

On the other hand six RING protein genes were found consistently up-regulated in high amylose line in all the four developmental stages (Fig. 7). Of these six genes, two genes (TraesCS4A02G112900, TraesCS1A02G341400) showed diferential log­ 2 FC > 2 and at the same time were found comparatively down regulated in low amylose line comparative to the parent (Group 2) (Fig. 8). Te RING protein gene TraesCS6B02G286500 was found to be extremely up-regulated at later seed developmental stages (21 and 28 DAA) in Group 1 (Fig. 8). Tese 3 RING protein genes were found up-regulated when amylose biosynthesis is high hence they might possibly act as positive regulators for amylose biosynthesis. At the same time expression of two key regulatory enzymes GBSSI and SBEIIa in starch biosynthesis was also analyzed. Te GBSSI was highly up-regulated and SBEIIa was highly down-regulated in high amylose line (Group 1). Whereas we found a vise-versa expression for the same (GBSSI and SBEIIa) in low amylose line (Group 2) (Fig. 8). Te higher and lower expression levels of GBSSI and SBEIIa in high amylose line might be the reason for high amylose starch biosynthesis. Te above identifed 11 candidate (eight down-regulated and three up- regulated) RING protein genes might be involved in regulation of GBSSI and SBEIIa via ubiquitin-mediated post-translational modifcations, either directly or through the modulation of other regulatory factors that has not been studied yet. As the RING proteins are involved in ubiquitin-mediated degradation pathway and can play a positive or negative role in regulation of a biosynthetic ­pathway44. So both the down regulation and up regula- tion of RING protein genes might be responsible for high amylose by targeting GBSSI and SBEIIa, respectively.

Correlation analysis of RING protein genes with starch pathway genes (Pearson’s correla‑ tion). Correlation between the normalized gene expression data of 36 RING protein genes and two starch pathway genes GBSSI and SBEIIa was analyzed. GBSSI and SBEIIa are key enzymes regulating the amylose and amylopectin biosynthesis respectively­ 7,9. Terefore, to explore the role of RING protein genes in starch biosyn- thesis, statistical correlation with key regulatory genes is essential. Te pairwise correlation analysis revealed that 55.5% (20) RING protein genes were negatively correlated with GBSSI. Out of twenty, only one gene (TraesC- S2D02G162600) showed very strong (r2 ≥ 0.80) and four genes (TraesCS5A02G049400, TraesCS6D02G254700, TraesCS6A02G274400, TraesCS3B02G000800) showed strong (r2 ≥ 0.60) signifcant negative correlation with

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 10 Vol:.(1234567890) www.nature.com/scientificreports/

Figure 7. Diferential gene expression (Log­ 2 fold change) data of 36 RING fnger protein genes in three genotypes ‘TAC 75’, ‘TAC 6’ and parent ‘C 306’ at four seed developmental stages (7, 14, 21, and 28 DAA (days afer anthesis) using qRT-PCR analysis. Diferential gene expression analysis was performed in two groups; (A) Group 1 (‘TAC 75’ vs. ‘C 306) and (B) Group 2 (‘TAC 6’ vs. ‘C 306’). ‘TAC 75’ and ‘TAC 6’ are high and low amylose mutant lines derived from parent variety ‘C 306’ with amylose percent ~ 65%, ~ 7%, ~ 26%, respectively. Te 11 candidate RING protein genes are marked with red color (down-regulated in high amylose line) and blue color (up-regulated in high amylose line).

GBSSI at p-value ≤ 0.05. While only two genes TraesCS4A02G112900 (r2 = 0.84) and TraesCS6B02G286500 (r2 = 0.78) showed signifcant positive correlation with GBSSI at p-value ≤ 0.05 (Fig. 9). Tese seven RING pro- tein genes, fve and two with strong negative and positive correlation might be involved in regulation of GBSSI expression respectively. Te correlation of 36 RING protein genes with SBEIIa showed 16 genes to be negatively correlated and 20 genes to be positively correlated (Fig. 9). Te correlation analysis revealed 4 genes (TraesCS4A02G11290, TraesCS6B02G286500, TraesCS4B02G164000 and TraesCS1A02G341400) showing signifcant strong negative correlation with SBEIIa (r2 ≥ 0.60). However, two genes (TraesCS2D02G162600, TraesCS3B02G000800) showed very strong (r2 ≥ 0.80) and other two genes (TraesCS6D02G254700, TraesCS3B02G461900) showed signifcant strong (r2 ≥ 0.60) positive correlation with SBEIIa at p-value ≤ 0.05. Tese eight RING protein genes, four each with strong negative and positive correlation to SBEIIa might be involved in the regulation of SBEIIa expression. From the above correlation analysis with GBSSI and SBEIIa, fve RING protein genes (out of 36) were found to have signifcant negative or positive correlation with both the genes, hence total 10 RING protein genes found to be correlated with GBSSI and SBEIIa (Fig. 9). Tese results suggest the possible involvement of these RING protein genes in regulation of both GBSSI and SBEIIa at protein levels, through the post-translational modifca- tions via direct or indirect gene regulation. Discussion Many genetic analyses have shown that large number of RING proteins have been identifed as positive or negative regulators of various biological processes. By considering their importance in plants, RING proteins were initially identifed in A. thaliana, encoding ~ 36% RING protein genes out of the ~ 1400 E3 ­ligases24,45,46. In the present study by exploring the hexaploid wheat genome (Triticum aestivum L., 2n = 6x = 42, AABBDD) we identifed 1272 potential RING domains in 1255RING fnger proteins (Table S1). Tese potential RING domains were further classifed in four major groups—RING-H2 (875), RING-HC (323), RING-v (67) and RING-G (7) (Fig. 1) illustrates that large number of identifed RING proteins 79% and 25.39% are RING-H2 and RING-HC types, respectively. Previous studies in A. thaliana have shown the presence of ~ 2% RING protein genes with eight types of RING domains- RING-H2 (241), RING-HCa (145), RING-HCb (41), RING-D (10), RING-G (1), RING-S/T (4), RING-v (25), and RING-C2 (10)24. We were unable to identify some above previously identifed RING domains from Arabidopsis and other plant species in wheat genome. We were also not able to diferentiate the identifed RING-HC group domains in RING-HCa and RING-HCb, all were found RING-HCa type as in rice­ 37. Our analysis of additional domains in wheat RING fnger proteins identifed many protein–protein inter- action domains, nucleic acid and ubiquitin binding domains in diferent organizations that have been previously identifed in Arabidopsis, apple, B. rapa, and B. oleracea24,36–39. Te 29.48% proteins had only RING domain, on the other hand 70.52% were having one or more additional domains other than RING (Supplementary Table S3). Te transmembrane domain was most frequently present with RING domain, indicating their role as membrane integral proteins. Copine and NB-ARC domain identifed in wheat RING domain containing proteins involved in lipid binding activities and nucleotide binding, ­respectively47,48. In wheat multiple domain of unknown function (DUFs), DUF1117, DUF4792, DUF4793, DUF1232, and DUF3675 were identifed rather than only DUF1117

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 11 Vol.:(0123456789) www.nature.com/scientificreports/

Figure 8. Bar graphs showing the diferential gene expression of 11 candidate RING protein genes along with GBSSI and SBEIIa using qRT-PCR for amylose biosynthesis at (A) 7 DAA, (B) 14 DAA, (C) 21 DAA and (D) 28 DAA in ‘TAC 75’ vs. ‘C 306’ and ‘TAC 6’ vs. ‘C 306’. ‘TAC 75’ and ‘TAC 6’ are high and low amylose mutant lines derived from parent variety ‘C 306’ with amylose percent ~ 65%, ~ 7%, ~ 26%, respectively. Te results were obtained using three technical and three biological replicates and are expressed as mean ± SD.

Figure 9. Pair wise Pearson’s correlation analysis of 10 RING protein genes with two starch pathway genes GBSSI and SBEIIa. Te signifcant values of correlation coefcient (r) labeled with *p ≤ 0. 05, **p ≤ 0. 01 and ***p ≤ 0. 001.

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 12 Vol:.(1234567890) www.nature.com/scientificreports/

found in Arabidopsis, most of the time DUF domain found in combination of transmembrane domains (Sup- plementary Table S3). Te presence of additional domains and their diferent organizations in RING proteins suggests the role in diferent biological processes that might be specifc for diferent organisms. Phylogenetic clustering of RING fnger domains clearly demonstrated their evolutionary relationship. All the four RING domains (RING-H2, RING-HC, RING-v, RING-G) clustered with their respective group domains with some exceptions. Te intermixing of a specifc domain with other might be the reason of variation in protein sequences except the conserved metal ligands, during evolutionary process. Tis study shows that in spite of diferent RING types they share some similarities that confer their same origin during evolution. To look more into the evolutionary history of RING domain proteins diferent orthologous RING proteins can be considered for phylogeny, this will give the clear view of their origin, structural and functional characteristics. Furthermore, our study focused to fnd out the genomic locations of RING protein genes and their gene duplication events within three homeologous genomes (A, B and D) in wheat. Wheat RING protein genes were mapped onto all 21 chromosomes. We observed that most of the identifed RING protein genes were located on 7D (6. 7%) and less distributed on 4B (3. 5%) chromosome (Supplementary Fig. S3). Wheat (Triticum aes- tivum) is an allohexaploid (2n = 6x = 42, genome AABBDD) originated by two isolated hybridization events 10, 000 years ago. Hence, wheat contains three genomes from closely related species, assumed having triplicate copies of genes followed by chromosomal doubling to maintain the fertility­ 49,50. We identifed 4 singleton (not duplicated) and 49, 15, 90 and 1080 RING protein genes as dispersed, proximal, tandem and WGD/segmental duplicated genes, respectively. Te RING protein genes on homeologous chromosome 3 and 7 were found whole genome/ segmental duplicated within their A, B, D genome, no collinearity from other chromosomes found. Te genes from homeologous chromosome 1 (1A, 1B and 1D) were found collinear within the chromosome and also showed collinearity in large extent with other homeologous chromosome 3 (3A, 3B, and 3D) (Fig. 3). Results of duplication events indicated that in the expansion of RING protein gene family in wheat whole genome duplication (WGD)/segmental duplications has played major role. Te analysis from RNA-seq data revealed that of 1255 RING protein genes, a set of 557 genes showed no expression at any of the three developmental stages, considered as possibly no role in seed development. Te 698 RING protein genes that were preferentially expressed at any seed developmental stage (2 DAA, 14 DAA and 28 DAA), suggest possible role in developing wheat seed. Te 43.84% genes were found to be expressed in all the above developmental stages, while, remaining revealed seed development specifc expression (Fig. 4). Te diferent expression patterns and expression levels of RING protein genes provide information of their functional diversity with diferent extent in various biological processes. If possible role of these RING proteins is determined, it will be great impact on seed quantity and quality control. Further, the 698 seed specifc RING protein genes were analyzed to illustrate the involvement in amylose biosynthesis. In seeds amylose is the main component for resistant starch, a healthy starch that is benefcial for combating gastrointestinal diseases, diabetes and obesity as well as play important role in food processing. Te expression analysis from in house transcriptome data of amylose variants and qRT-PCR data of 36 wheat RING protein genes revealed that RING protein genes were expressed at both initial and late developmental stages, suggesting their putative role throughout the seed development (Supplementary Table S8, Fig. 7). Diferential gene expression analysis of 36 RING protein genes in amylose variant genotypes also revealed 8 strongly down- regulated and 3 up-regulated genes in high amylose line. Tese RING protein genes might play potential role as positive or negative regulators of amylose biosynthesis by acting as efective E3 ligases in ubiquitin-mediated post-translational modifcations. As ubiquitin-mediated post-translational modifcations are involved in vari- ous biological processes including stress­ 51, ­growth28, ­immunity52, ­photosynthesis53 and hormone signaling­ 54, but their direct role in starch biosynthesis is not yet known. A study in wheat has been shown the role of TaGW2, a RING E3 ligase in seed size and weight. Tey hypothesized the increased seed weight might be a reason of higher accumulation of starch controlled by ­TaGW233. Tis study suggests only the probable role of RING proteins in starch biosynthesis that need to be explored. Te correlation studies of 36 RING protein genes were performed with GBSSI and SBEIIa, two key enzymes regulating the amylose and amylopectin biosynthesis, respectively­ 7,9. Terefore, to explore the role of RING proteins in starch biosynthetic pathway, statistical correlation with key regulatory starch pathway genes was essential. Te correlation studies of 36 RING protein genes with GBSSI and SBEIIa expression data showed 10 strongly correlated RING protein genes, with GBSSI and SBEIIa. Tese results indicate the involvement of RING protein genes in the regulation of transcripts levels and further pro- tein levels of GBSSI and SBEIIa, hence will possibly impact on the amylose biosynthesis via direct regulation or through negative and positive regulators of these enzymes. Tis study has provided substantial information of RING protein genes, their correlation with starch biosynthetic genes and potential targets for wheat amylose regulation in wheat. Conclusion In the present study, we identifed 1255 RING proteins in wheat through in silico approaches. Te RING protein genes were found to be distributed all over wheat genome with duplication across the genome. Majority of vari- ations and expansion of RING protein gene family in wheat took place through several duplication events that has contributed for the functional diversifcation of RING proteins. Te expression analysis of 698 RING protein genes during various seed developmental stages revealed their possible involvement in seed development. Te identifed mutation in RING protein genes could be the reason of amylose variation in wheat mutants. Interest- ingly, this study demonstrated that RING E3 ligases might play a potential role in the amylose biosynthesis as positive or negative regulators, thus imparting great knowledge for the grain quality enhancement. Tis whole study would be helpful to reduce the study gap of ubiquitin-mediated post translational modifcations of amylose regulatory enzymes and other seed functional traits.

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 13 Vol.:(0123456789) www.nature.com/scientificreports/

Materials and methods Plant materials. Two mutant lines (M6 generation), ‘TAC 75’ (amylose content ~ 65%) and ‘TAC 6’ (amyl- ose content ~ 7%) developed via EMS mutagenesis along with their parent wheat variety, ‘C 306’ (amylose con- tent ~ 26%) were used in this ­study10.

Genome‑wide identifcation of RING domain containing proteins in wheat. To identify pre- sumptive wheat RING fnger domain containing proteins, two strategies were followed. In the frst strategy RING fnger proteins of Oryza sativa37 and A. thaliana24 were taken as query data set to identify potential wheat orthologs using Ensembl T. aestivum protein database (RefSeq 1.1) (https://​plants.​ensem​bl.​org/​Triti​cum_​aesti​ vum/​Info/​Index). Te second strategy used to identify proteins containing RING [ZnF-RING (IPR001841)] and Pfam domain [zf-C3HC4 (PF00097)] of InterProScan against the same T. aestivum protein database­ 55. To confrm the presence of RING domains in the retrieved irredundant protein sequences from both the strate- gies, all the sequences were analyzed by Single Modular Architecture Research Tool (SMART) database (http://​ smart.​embl-​heide​lberg.​de/) integrated with the Pfam domain database. Further, the confrmation of RING pro- teins was done by manual inspection of each protein sequences for the presence of any of the eight conserved zinc coordinating metal ligands (Cys or His) and the distance between each metal ligand. Te identifed RING domain containing proteins were classifed into groups based on the amino acid residues at metal ligand posi- tions and the residues number between each metal ­ligand24. Proteins confrmed by SMART database but lack- ing one or more metal ligands were not considered in any group and they were classifed as incomplete RING domain containing proteins. Sequence logos of multiple domain alignments from each representing group were created using online WebLogo tool (https://​weblo​go.​berke​ley.​edu/​logo.​cgi).

Multiple sequence alignment and phylogenetic analysis of RING domains. Multiple sequence alignment was performed on the sequences of the identifed RING domain that were extracted from their respec- tive protein sequences in Clustal X (Version 2.1; http://​www.​clust​al.​org/​clust​al2/). Te parameters for pairwise alignment were 40 gap opening and 0.8 gap extension penalty using PAM350 protein matrix and other criteria at default setting. Further, the aligned RING domain sequences were manually edited in BioEdit sofware to cor- rectly align eight metal ligand residues and were used for performing phylogenetic analysis by MEGA X sofware (Version 10.1). Phylogenetic tree was generated by Neighbor-Joining (NJ) algorithm with 1000 bootstrap for signifcant evaluation of phylogenetic tree. Evolutionary distances were inferred using Jones–Taylor–Tornton (JTT) model and for gap profling pairwise gap deletion model was used.

Identifcation of additional RING domains. To identify the presence of other possible domains in RING fnger proteins, SMART database considering Pfam domains and signal peptide was used. RING contain- ing proteins were classifed into diferent groups based on the presence or absence and organization of the iden- tifed additional domains. Species with the same protein domain architectures as that in wheat RING domain containing proteins were identifed using NCBI BLASTp search in model organisms only.

Chromosomal location of wheat RING protein genes. Te chromosomal locations of wheat RING protein genes were retrieved from GFF fle of T. aestivum genome database downloaded from Ensembl plants (fp://​fp.​ensem​blgen​omes.​org/​pub/​plants/​relea​se-​46/​gf3/​triti​cum_​aesti​vum). Te genes that were not mapped on any chromosome were excluded from the analysis. Chromosomal location of genes was drawn onto T. aesti- vum 21 chromosomes by Circos sofware (Version 0. 69-9). All duplication events such as dispersed, proximal, tandem, whole genome (WGD)/segmental and singleton (not duplicated) in RING protein genes were predicted by MCScanX tool­ 56. WGD/segmental duplications are shown in comparative analysis of synteny of RING pro- tein genes on the entire wheat chromosomes using Circos sofware.

Expression analysis of RING containing genes in developing wheat seeds. Te expression pro- fles of wheat RING containing genes were analyzed at three seed developmental stages (2, 14, and 30 DAA) in the previously reported RNA-seq data­ 57 using RefSeq1.1 nucleotide database in wheat expression browser (http://​www.​wheat-​expre​ssion.​com/). Normalized transcripts per million (TPM) values were used to analyze the expression pattern at diferent developmental stages. Clustered heat map showing expression profle of wheat RING containing genes was generated by MeV sofware­ 58. Te hierarchical clustering was performed using uncentered pearson correlation distance matric and complete linkage clustering method. Additionally, the diferential gene expression of RING protein genes was also analyzed using the in-house transcriptome data of mutant lines (‘TAC 75’ and ‘TAC 6’) and parent (‘C 306’) in three biological replicates. For the whole transcrip- tome data analysis CLC genomics workbench (CLC GWB, Version 20) was used. High-quality paired end reads were aligned against annotated wheat genome reference assembly (IWGSC RefSeq1.0). Diferential expression analysis of genes was performed using normalized RPKM (reads per kilobase of exon model per million reads) values and genes presenting p-value ≤ 0.05 were considered as signifcant and were further used for downstream analysis. Te diferential gene expression values of RING containing genes were acquired by DESeq2 (v1.22.1) and genes with fold change > 2 were retained for further analysis. Te RING containing genes were selected based on expression values from the public domain and in-house transcriptome data and were used for qRT- PCR validation in mutant lines along with parent.

RING protein genes variant identifcation in mutant lines. For variant analysis of RING protein genes in mutants ‘TAC 75’ and ‘TAC 6’ the Illumina generated reads were used and high quality (Q30) reads

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 14 Vol:.(1234567890) www.nature.com/scientificreports/

were assembled and mapped against the wheat reference genome (Ensemble release 49) using the BWA-MEM tool (version 0.7.17) [17, Paolacci et al.59)]. Tis alignment fle (.bam) was further used for variant identifcation by Samtools using parameters described by Paolacci et al.59. Genetic variants like SNPs, insertions, deletions and MNPs were annotated using SnpEf (version 4.3t) tool­ 25 against the wheat reference genome. Te in-house Perl scripts were used to analyse the distribution of variants (SNPs and Indels) in mutants ‘TAC 6’ and ‘TAC 75’ among identifed 698 RING protein genes possibly involved in grain development.

qRT‑PCR validation of candidate RING protein genes during seed development. Genome-spe- cifc primers of candidate RING protein genes, limiting factor genes of starch biosynthesis (GBSSI and SBEIIa)10, and wheat ADP-Ribosylation Factor (ARF) as an internal control­ 60 were designed using OligoCalc (http://​bioto​ ols.​nubic.​north​weste​rn.​edu/​Oligo​Calc.​html). RNAs extracted from primary individual spikes from three bio- logical replicate samples were used for qRT-PCR analysis. Spikes were tagged on the frst day of anthesis and harvested at 7, 14, 21, and 28 day afer anthesis (DAA), frozen in liquid nitrogen, and stored at − 80 °C till fur- ther use. RNA was isolated by introducing minor changes in Trizol ­method20 and cDNA was synthesized using iScriptgDNA clear cDNA synthesis kit (Bio-Rad, USA). Tree biological replicates with their three technical replicates were used for qRT-PCR analysis using 1:10 diluted cDNA and Fast SYBR Green Master Mix as per manufacturer’s instruction in 7500 Fast Real-Time PCR System.

Statistical analysis. To analyze the relationship among wheat RING protein genes and key regulatory genes of starch metabolism (GBSSI, SBEIIa), pair wise Pearson’s correlation coefcient (r) and their signifcance test was performed using Graph Pad Prism (version 5) with p-value criteria ≤ 0.05. Correlation analysis was done using their mean normalized Ct values (at four diferent seed developmental stages).

Ethics declarations. All experiments were performed in accordance with relevant institutional guidelines.

Received: 27 October 2020; Accepted: 10 May 2021

References 1. Gao, S., Wang, W., Guo, T. & Han, J. C-N metabolic characteristics in fag leaf and starch accumulating developments in seed during grain flling stage in two winter wheat cultivars with diferent spike type. Acta Agron. Sin. 1, 1–10 (2003). 2. Tester, R. F., Karkalas, J. & Qi, X. Starch structure and digestibility enzyme-substrate relationship. World’s Poult. Sci. J. https://doi.​ ​ org/​10.​1079/​WPS20​040014 (2004). 3. Brouns, F., Kettlitz, B. & Arrigoni, E. Resistant starch and ‘the butyrate revolution’. Trends Food Sci. Technol. https://​doi.​org/​10.​ 1016/​S0924-​2244(02)​00131-0 (2002). 4. Van Hung, P., Maeda, T. & Morita, N. Waxy and high-amylose wheat starches and fours-characteristics, functionality and applica- tion. Trends Food Sci. Technol. https://​doi.​org/​10.​1016/j.​tifs.​2005.​12.​006 (2006). 5. Slade, A. J. et al. Development of high amylose wheat through TILLING. BMC Plant Biol. https://doi.​ org/​ 10.​ 1186/​ 1471-​ 2229-​ 12-​ 69​ (2012). 6. Kumar, P. et al. Pivotal role of bZIPs in amylose biosynthesis by genome survey and transcriptome analysis in wheat (Triticum aestivum L.) mutants. Sci. Rep. 8, 1–15. https://​doi.​org/​10.​1038/​s41598-​018-​35366-8 (2018). 7. Regina, A. et al. High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats. Proc. Natl. Acad. Sci. USA. https://​doi.​org/​10.​1073/​pnas.​05107​37103 (2006). 8. Wang, Z., Li, W., Qi, J., Shi, P. & Yin, Y. Starch accumulation, activities of key enzyme and gene expression in starch synthesis of wheat endosperm with diferent starch contents. J. Food Sci. Technol. https://​doi.​org/​10.​1007/​s13197-​011-​0520-z (2014). 9. Sestili, F. et al. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes. BMC Plant Biol. https://doi.​ ​ org/​10.​1186/​1471-​2229-​10-​144 (2010). 10. Mishra, A., Singh, A., Sharma, M., Kumar, P. & Roy, J. Development of EMS-induced mutation population for amylose and resist- ant starch variation in bread wheat (Triticum aestivum) and identifcation of candidate genes responsible for amylose variation. BMC Plant Biol. https://​doi.​org/​10.​1186/​s12870-​016-​0896-z (2016). 11. Sehnke, P. C., Chung, H. J., Wu, K. & Ferl, R. J. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proc. Natl. Acad. Sci. USA. https://​doi.​org/​10.​1073/​pnas.​98.2.​765 (2001). 12. Hendriks, J. H. M., Kolbe, A., Gibon, Y., Stitt, M. & Geigenberger, P. ADP-glucose pyrophosphorylase is activated by posttransla- tional redox-modifcation in response to light and to sugars in leaves of arabidopsis and other plant species. Plant Physiol. https://​ doi.​org/​10.​1104/​pp.​103.​024513 (2003). 13. Tetlow, I. J. et al. Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein–protein interac- tions. Plant Cell https://​doi.​org/​10.​1105/​tpc.​017400 (2004). 14. Hershko, A. Te ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Difer. https://​doi.​org/​10.​1038/​sj.​cdd.​44017​02 (2005). 15. McNeilly, D., Schofeld, A. & Stone, S. L. Degradation of the stress-responsive enzyme formate dehydrogenase by the RING-type E3 ligase Keep on Going and the ubiquitin 26S proteasome system. Plant Mol. Biol. https://​doi.​org/​10.​1007/​s11103-​017-​0691-8 (2018). 16. Hershko, A. & Ciechanover, A. Te ubiquitin system for protein degradation. Annu. Rev. Biochem. https://doi.​ org/​ 10.​ 1146/​ annur​ ​ ev.​bi.​61.​070192.​003553 (1992). 17. Seol, J. H. et al. Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF defne a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev. https://​doi.​org/​10.​1101/​gad.​13.​12.​1614 (1999). 18. Moon, J., Parry, G. & Estelle, M. Te ubiquitin-proteasome pathway and plant development. Plant Cell https://​doi.​org/​10.​1105/​ tpc.​104.​161220 (2004). 19. Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. https://​doi.​org/​10.​1146/​annur​ev.​bioch​em.​70.1.​503 (2001). 20. Lorick, K. L. et al. RING fngers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. USA. https://​doi.​org/​10.​1073/​pnas.​96.​20.​11364 (1999).

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 15 Vol.:(0123456789) www.nature.com/scientificreports/

21. Zheng, N. et al. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature https://doi.​ org/​ 10.​ 1038/​ 41670​ ​ 3a (2002). 22. Furukawa, M., He, Y. J., Borchers, C. & Xiong, Y. Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat. Cell Biol. https://​doi.​org/​10.​1038/​ncb10​56 (2003). 23. Kosarev, P., Mayer, K. F. X. & Hardtke, C. S. Evaluation and classifcation of RING-fnger domains encoded by the Arabidopsis genome. Genome Biol. https://​doi.​org/​10.​1186/​gb-​2002-3-​4-​resea​rch00​16 (2002). 24. Stone, S. L. et al. Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol. https://​doi.​org/​10.​ 1104/​pp.​104.​052423 (2005). 25. McNellis, T. W. et al. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains. Plant Cell https://​doi.​org/​10.​1105/​tpc.6.​4.​487 (1994). 26. Liu, H. et al. Te RING-Type E3 Ligase XBAT35.2 is involved in cell death induction and pathogen response. Plant Physiol. https://​ doi.​org/​10.​1104/​pp.​17.​01071 (2017). 27. Dong, C. H., Agarwal, M., Zhang, Y., Xie, Q. & Zhu, J. K. Te negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc. Natl. Acad. Sci. USA. https://​doi.​org/​10.​1073/​pnas.​06028​74103 (2006). 28. Stone, S. L., Williams, L. A., Farmer, L. M., Vierstra, R. D. & Callis, J. KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell https://​doi.​org/​10.​1105/​tpc.​106.​046532 (2006). 29. Lim, C. W., Baek, W. & Lee, S. C. Te pepper RING-type E3 ligase CaAIRF1 regulates ABA and drought signaling via CaADIP1 protein phosphatase degradation. Plant Physiol. https://​doi.​org/​10.​1104/​pp.​16.​01817 (2017). 30. Serrano, M. & Guzmán, P. Isolation and gene expression analysis of Arabidopsis thaliana mutants with constitutive expression of ATL2, an early elicitor-response RING-H2 zinc-fnger gene. Genetics https://​doi.​org/​10.​1534/​genet​ics.​104.​028043 (2004). 31. Ling, Q., Huang, W., Baldwin, A. & Jarvis, P. Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science https://​doi.​org/​10.​1126/​scien​ce.​12250​53 (2012). 32. Song, X. J., Huang, W., Shi, M., Zhu, M. Z. & Lin, H. X. A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nat. Genet. https://​doi.​org/​10.​1038/​ng2014 (2007). 33. Bednarek, J. et al. Down-regulation of the TaGW2 gene by RNA interference results in decreased grain size and weight in wheat. J. Exp. Bot. https://​doi.​org/​10.​1093/​jxb/​ers249 (2012). 34. Geng, J. et al. TaGW2-6A allelic variation contributes to grain size possibly by regulating the expression of cytokinins and starch- related genes in wheat. Planta https://​doi.​org/​10.​1007/​s00425-​017-​2759-8 (2017). 35. Sestili, F. et al. Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes. Teor. Appl. Genet. https://​doi.​org/​ 10.​1007/​s00122-​018-​3229-9 (2019). 36. Li, H. et al. PERSISTENT TAPETAL CELL1 encodes a PHD-fnger protein that is required for tapetal cell death and pollen devel- opment in rice. Plant Physiol. https://​doi.​org/​10.​1104/​pp.​111.​175760 (2011). 37. Lim, S. D. et al. A gene family encoding RING fnger proteins in rice: Teir expansion, expression diversity, and co-expressed genes. Plant Mol. Biol. https://​doi.​org/​10.​1007/​s11103-​009-​9576-9 (2010). 38. Alam, I. et al. Genome-wide identifcation, evolution and expression analysis of RING fnger protein genes in Brassica rapa. Sci. Rep. https://​doi.​org/​10.​1038/​srep4​0690 (2017). 39. Yang, Y. Q., Lu, Y. H. & King, J. Genome-wide survey, characterization, and expression analysis of RING fnger protein genes in Brassica oleracea and their syntenic comparison to Brassica rapa and Arabidopsis thaliana. Genome https://​doi.​org/​10.​1139/​gen-​ 2018-​0046 (2018). 40. Buljan, M. & Bateman, A. G. Te evolution of protein domain families. Biochem. Soc. Trans. https://doi.​ org/​ 10.​ 1042/​ BST03​ 70751​ (2009). 41. Yuan, X. et al. Global analysis of ankyrin repeat domain C3HC4-type RING fnger gene family in plants. PLoS ONE https://​doi.​ org/​10.​1371/​journ​al.​pone.​00580​03 (2013). 42. Whittaker, C. A. & Hynes, R. O. Distribution and evolution of von Willebrand/integrin A domains: Widely dispersed domains with roles in cell adhesion and elsewhere. Mol. Biol. Cell https://​doi.​org/​10.​1091/​mbc.​E02-​05-​0259 (2002). 43. Mishra, A. K., Puranik, S. & Prasad, M. Structure and regulatory networks of WD40 protein in plants. J. Plant Biochem. Biotechnol. https://​doi.​org/​10.​1007/​s13562-​012-​0134-1 (2012). 44. Duplan, V. & Rivas, S. E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2014.​00042 (2014). 45. Vierstra, R. D. Te ubiquitin-26S proteasome system at the nexus of plant biology. Nat. Rev. Mol. Cell Biol. https://doi.​ org/​ 10.​ 1038/​ ​ nrm26​88 (2009). 46. Kraf, E. et al. Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol. https://​doi.​org/​10.​1104/​pp.​105.​067983 (2005). 47. Tomsig, J. L. & Creutz, C. E. Copines: A ubiquitous family of Ca2+-dependent phospholipid-binding proteins. Cell. Mol. Life Sci. https://​doi.​org/​10.​1007/​s00018-​002-​8522-7 (2002). 48. Tameling, W. I. L. et al. Te tomato R gene products i–2 and Mi-1 are functional ATP binding proteins with ATPase activity. Plant Cell https://​doi.​org/​10.​1105/​tpc.​005793 (2002). 49. Wang, J. et al. Variability of gene expression afer polyhaploidization in wheat (Triticum aestivum L.). G3 https://doi.​ org/​ 10.​ 1534/​ ​ g3.​111.​000091 (2011). 50. Huo, N. et al. Gene duplication and evolution dynamics in the homeologous regions harboring multiple prolamin and resistance gene families in hexaploid wheat. Front. Plant Sci. https://​doi.​org/​10.​3389/​fpls.​2018.​00673 (2018). 51. Lee, H. K. et al. Drought stress-induced Rma1H1, a RING membrane-anchor E3 ubiquitin ligase homolog, regulates aquaporin levels via ubiquitination in transgenic arabidopsis plants. Plant Cell https://​doi.​org/​10.​1105/​tpc.​108.​061994 (2009). 52. Zhang, L., Du, L., Shen, C., Yang, Y. & Poovaiah, B. W. Regulation of plant immunity through ubiquitin-mediated modulation of Ca2+-calmodulin-AtSR1/CAMTA3 signaling. Plant J. https://​doi.​org/​10.​1111/​tpj.​12473 (2014). 53. Agetsuma, M., Furumoto, T., Yanagisawa, S. & Izui, K. Te ubiquitin-proteasome pathway is involved in rapid degradation of phosphoenolpyruvate carboxylase kinase for C4 photosynthesis. Plant Cell Physiol. https://​doi.​org/​10.​1093/​pcp/​pci043 (2005). 54. Kelley, D. R. & Estelle, M. Ubiquitin-mediated control of plant hormone signaling. Plant Physiol. https://​doi.​org/​10.​1104/​pp.​112.​ 200527 (2012). 55. Jones, P. et al. InterProScan 5: Genome-scale protein function classifcation. Bioinformatics https://​doi.​org/​10.​1093/​bioin​forma​ tics/​btu031 (2014). 56. Wang, Y. et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. https://​doi.​org/​10.​1093/​nar/​gkr12​93 (2012). 57. Choulet, F. et al. Structural and functional partitioning of bread wheat chromosome 3B. Science https://​doi.​org/​10.​1126/​scien​ce.​ 12497​21 (2014). 58. Howe, E. A., Sinha, R., Schlauch, D. & Quackenbush, J. RNA-Seq analysis in MeV. Bioinformatics https://​doi.​org/​10.​1093/​bioin​ forma​tics/​btr490 (2011). 59. Paolacci, A. R., Tanzarella, O. A., Porceddu, E. & Ciaf, M. Identifcation and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol. Biol. https://​doi.​org/​10.​1186/​1471-​2199-​10-​11 (2009).

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 16 Vol:.(1234567890) www.nature.com/scientificreports/

60. Rio, D. C., Ares, M., Hannon, G. J. & Nilsen, T. W. Purifcation of RNA using TRIzol (TRI Reagent). Cold Spring Harb. Protoc. https://​doi.​org/​10.​1101/​pdb.​prot5​439 (2010). 61. Bork, P. Hundreds of ankyrin-like repeats in functionally diverse proteins: Mobile modules that cross phyla horizontally?. Proteins Struct. Funct. Bioinform. https://​doi.​org/​10.​1002/​prot.​34017​0405 (1993). 62. Li, S. et al. Identifcation of a novel cytoplasmic protein that specifcally binds to nuclear localization signal motifs. J. Biol. Chem. https://​doi.​org/​10.​1074/​jbc.​273.​11.​6183 (1998). 63. Tomsig, J. L. & Creutz, C. E. Copines: A ubiquitous family of Ca 2+-dependent phospholipid-binding proteins. Cell. Mol. Life Sci. https://​doi.​org/​10.​1007/​s00018-​002-​8522-7 (2002). 64. Caruthers, J. M., Johnson, E. R. & McKay, D. B. Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. Proc. Natl. Acad. Sci. USA https://​doi.​org/​10.​1073/​pnas.​97.​24.​13080 (2000). 65. Iyer, L. M., Babu, M. M. & Aravind, L. Te HIRAN domain and recruitment of chromatin remodeling and repair activities to damaged DNA. Cell Cycle https://​doi.​org/​10.​4161/​cc.5.​7.​2629 (2006). 66. Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H. & Hunter, T. Te PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. Mol. Cell https://​doi.​org/​10.​1016/​S1097-​2765(02)​00519-1 (2002). 67. van der Biezen, E. A. & Jones, J. D. Te NB-ARC domain: A novel signalling motif shared by plant resistance gene products and regulators of cell death in animals. Curr. Biol. https://​doi.​org/​10.​1016/​s0960-​9822(98)​70145-9 (1998). 68. Van Dyck, L., Pearce, D. A. & Sherman, F. PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochon- drial function in the yeast Saccharomyces cerevisiae. J. Biol. Chem. https://​doi.​org/​10.​1016/​s0021-​9258(17)​42340-4 (1994). 69. Sheng, Y. et al. Molecular basis of Pirh2-mediated p53 ubiquitylation. Nat. Struct. Mol. Biol. https://​doi.​org/​10.​1038/​nsmb.​1521 (2008). 70. Leng, R. P. et al. Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation. Cell https://​doi.​org/​10.​1016/​S0092-​ 8674(03)​00193-4 (2003). 71. Worthington, M. T., Amann, B. T., Nathans, D. & Berg, J. M. Metal binding properties and secondary structure of the zinc-binding domain of Nup475. Proc. Natl. Acad. Sci. USA https://​doi.​org/​10.​1073/​pnas.​93.​24.​13754 (1996). 72. Ponting, C. P., Blake, D. J., Davies, K. E., Kendrick-Jones, J. & Winder, S. J. ZZ and TAZ: New putative zinc fngers in dystrophin and other proteins. Trends Biochem. Sci. https://​doi.​org/​10.​1016/​S0968-​0004(06)​80020-4 (1996). 73. Bürglin, T. R. Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif. BMC Genom.s https://​doi.​org/​10.​1186/​1471-​2164-9-​127 (2008). 74. Kang, R. S. et al. Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding. Cell https://​doi.​ org/​10.​1016/​S0092-​8674(03)​00362-3 (2003). 75. Yafe, M. B. & Elia, A. E. H. Phosphoserine/threonine-binding domains. Curr. Opin. Cell Biol. https://​doi.​org/​10.​1016/​S0955-​ 0674(00)​00189-7 (2001). 76. Zhang, B. et al. GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth. Cell Res. https://doi.​ org/​ 10.​ ​ 1038/​cr.​2008.​75 (2008). Acknowledgements AP is thankful to National Agri-Food Biotechnology Institute (NABI), an autonomous institute of Department of Biotechnology (DBT), Government of India (GOI) for providing research facility and fnancial support to carry out research work. We also acknowledge DeLCON (DBT-electronic library consortium), Gurugram, India, for the online journal access. Author contributions J.R.: conceptualization and supervision of the experiment. A.P.: wrote the original draf paper, performed experi- mental work, data analysis, validation, and visualization. M.S.R.: wrote a draf paper and contributed to the analysis. A.S.: data analysis. A.M.: Interpretation of data and drafing the paper. Pr.K. and V.F.: editing and refning the manuscript. Pa.K. and A.B.: contributed in the experimental work and analysis. S.K.V.: editing and refning the manuscript. All authors reviewed the manuscript.

Competing interests Te authors declare no competing interests. Additional information Supplementary Information Te online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​021-​90685-7. Correspondence and requests for materials should be addressed to J.R. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations. Open Access Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

© Te Author(s) 2021

Scientifc Reports | (2021) 11:11461 | https://doi.org/10.1038/s41598-021-90685-7 17 Vol.:(0123456789)